Genetic complementation by cloned bacteriophage T4 late genes
Genetic complementation by cloned bacteriophage T4 late genes
复制标题
克隆噬菌体 T4 晚期基因的遗传互补
作者：
K. Jacobs;L. M. Albright;D. Shibata;E. Geiduschek
含有晚期基因无义突变的T4噬菌体被发现由位于质粒或噬菌体载体上的4个共轭T4基因(7、11、23或24)在遗传上互补。互补作用处于非常低的水平，除非感染噬菌体携带denB突变(这会取消T4DNA内切酶IV的活性)。在大多数实验中，感染噬菌体还存在DENA突变，该突变会取消T4DNA内切酶II的活性。Alc/unf基因的突变(允许表达含有dcMP的T4晚期基因)进一步提高了互补效率。用于这些实验的大多数alc/unf突变噬菌体菌株都是构建成包含基因56突变的，该突变阻止dCTP分解，并允许复制产生含有dcMP的T4DNA。Alc/unf：56突变组合对互补效率的影响因T4晚期基因的不同而不同。尽管克隆的T4基因与感染基因组之间有2-14kb的同源性，但T4 DEN：Alc噬菌体感染后重组率普遍较低(基因23中的两个突变体较高，基因7和基因11中的突变体较低)。更重要的是，当基因23的互补必须在重组之前进行时，互补的效率大大降低。我们的结论是，克隆的T4晚期基因的高互补效率不需要依赖于先前的完整断裂-重组事件，这些事件将这些基因从驻留的质粒转位到感染T4基因组上的晚期启动子。原核表达载体上的完整基因23降低了T4噬菌体的产量。这种负互补效应的大小在不同的质粒中是不同的；在极端情况下(PLA3)，产量几乎减少了10倍。因此，通过整合一部分病毒基因组，这些细胞可以说是部分不允许这种裂解病毒的细胞。
Bacteriophage T4 containing nonsense mutations in late genes was found to be genetically complemented by four conjugate T4 genes (7, 11, 23, or 24) located on plasmid or phage vectors. Complementation was at a very low level unless the infecting phage carried a denB mutation (which abolishes T4 DNA endonuclease IV activity). In most experiments, the infecting phage also had a denA mutation, which abolishes T4 DNA endonuclease II activity. Mutations in the alc/unf gene (which allow dCMP-containing T4 late genes to be expressed) further increased complementation efficiency. Most of the alc/unf mutant phage strains used for these experiments were constructed to incorporate a gene 56 mutation, which blocks dCTP breakdown and allows replication to generate dCMP-containing T4 DNA. Effects of the alc/unf:56 mutant combination on complementation efficiency varied among the different T4 late genes. Despite regions of homology, ranging from 2 to 14 kilobase pairs, between cloned T4 genes and infecting genomes, the rate of formation of recombinants after T4 den:alc phage infection was generally low (higher for two mutants in gene 23, lower for mutants in gene 7 and 11). More significantly, when gene 23 complementation had to be preceded by recombination, the complementation efficiency was drastically reduced. We conclude that high complementation efficiency of cloned T4 late genes need not depend on prior complete breakage-reunion events which transpose those genes from the resident plasmid to a late promoter on the infecting T4 genome. The presence of the intact gene 23 on plasmids reduced the yield of T4 phage. The magnitude of this negative complementation effect varied in different plasmids; in the extreme case (plasmid pLA3), an almost 10-fold reduction of yield was observed. The cells can thus be said to have been made partly nonpermissive for this lytic virus by incorporating a part of the viral genome.